Research Article

Construction of recombinant adenoviruses carrying the optimal shRNA template against goat PTHrP and successful suppression of PTHrP expression in mammary epithelial cells

Published: September 04, 2013
Genet. Mol. Res. 12 (3) : 3363-3374 DOI: 10.4238/2013.September.4.2

Abstract

Parathyroid hormone-related protein (PTHrP) is a protein member of the parathyroid hormone family that regulates the dynamic balance between blood and bone calcium during lactation. However, the mechanism of its regulation is not very clear. In order to establish a framework for further functional studies of the PTHrP gene in goat mammary gland epithelial cells during the lactation period, PTHrP cDNA was isolated from Xinong Saanen dairy goats. Its coding sequence is 534 bp in size. We also designed a short hairpin RNA (shRNA) to efficiently inhibit PTHrP expression and constructed recombinant adenoviruses carrying a template encoding this shRNA (AD-PTHrP-322) using the Block-iT shRNA interference system. Finally, the inhibition of PTHrP expression by the recombinant adenoviruses was confirmed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and Western blotting. qRT-PCR results showed that the expression of PTHrP mRNA in mammary epithelial cells was downregulated by 29.2, 68.1, and 82.6% 24, 48, and 72 h after the cells were infected with AD-PTHrP-322, respectively. Western blotting also showed that the expression of PTHrP was reduced in a time-dependent manner. These results suggest that AD-PTHrP-322 significantly inhibits the expression of PTHrP.

Parathyroid hormone-related protein (PTHrP) is a protein member of the parathyroid hormone family that regulates the dynamic balance between blood and bone calcium during lactation. However, the mechanism of its regulation is not very clear. In order to establish a framework for further functional studies of the PTHrP gene in goat mammary gland epithelial cells during the lactation period, PTHrP cDNA was isolated from Xinong Saanen dairy goats. Its coding sequence is 534 bp in size. We also designed a short hairpin RNA (shRNA) to efficiently inhibit PTHrP expression and constructed recombinant adenoviruses carrying a template encoding this shRNA (AD-PTHrP-322) using the Block-iT shRNA interference system. Finally, the inhibition of PTHrP expression by the recombinant adenoviruses was confirmed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and Western blotting. qRT-PCR results showed that the expression of PTHrP mRNA in mammary epithelial cells was downregulated by 29.2, 68.1, and 82.6% 24, 48, and 72 h after the cells were infected with AD-PTHrP-322, respectively. Western blotting also showed that the expression of PTHrP was reduced in a time-dependent manner. These results suggest that AD-PTHrP-322 significantly inhibits the expression of PTHrP.